Phenylketonuria is a human genetic disorder commonly known as classical PKU, which is a birth defect that is caused by an inborn in error amino acid metabolism. The condition is autosomal recessive and is characterized by the absence of the hepatic enzyme phenyl-alanine hydroxylase. This enzyme catalyses the hydroxylation of phenylalanine to tyrosine and the lack of activity causes severe mental retardation in untreated children. The prevalance of this hereditary disorder among caucaseans ranges from 1/5,400 in Ireland to 1/1,600 in Switzerland, with an average of about 1/8,000 in the United States. It has been estimated that 2 percent of the population are heterozygous carriers of this genetic disorder. Neonatal screening of all new-born infants for PKU is mandated by law in many Western countries including the United States since the disorder can be treated with limited success by long-term dietary correction. There is however no available means for prevention of the disorder due to the lack of reliable analytical methodologies for identification of heterozygous adults and homozygous recessive fetuses. Since amino acid sequence of the enzyme has not been determined, whether there are amino acid substitutions between the normal and PKU proteins are not known at the present time. Using Recombinant DNA Technology, we propose to isolate and characterize the human phenylalanine hydroxylase gene from normal and deficient individuals by molecular cloning. Comparison of the structural organization and nucleotide sequence between the cloned genes should reveal any amino acid substitutions in the variant proteins and would thereby establish the molecular basis of the deficiency at the gene level. This information will then permit the development of a simple and reliable method for heterozygote identification and prenatal diagnosis of the genetic disorder by gene mapping. Early detection of individuals with the genetic trait and prenatal diagnosis will promote prevention of the deficiency through genetic counceling. Finally, attempts will be made to better understand the cause(s) of the deficiency by examining the expression of the deficient genes after their introduction through DNA-mediated gene transfer into human cells in culture.